Human growth hormone (abbreviated to "hGH") is a single chain polypeptide hormone, the naturally occurring type of which consists of 191 amino acid residues. hGH usually occurs in a biologically active monomer form, but is known to aggregate into dimers and then polymers under thermal stress or mechanical stress such as shaking imposed on its pharmaceutical preparation, leading to a loss of its biological activity (Becker, G. W. et al.(1987) Biotechnol. Appl. Biochem.,9, p.478).
On the other hand, it is known that long-term storage of a hGH aqueous solution causes a gradual production of a deamidation products while less forming polymerization products of hGH. The deamidated hGH, although having no alteration in its biological activity (Becker,G. W. et al.(1988) Biotechnol. Appl. Biochem., 10, p.326), is undesirable in a pharmaceutical product as its presence is thought to imply declining qualities, and its allowable content is thus usually provided by the specification.
It is also generally known that the denaturation of hGH by aggregation occurs mainly under physical stresses, while its denaturation by deamidation occurs mainly under chemical stresses.
Due to these problems, an optimal aqueous preparation of hGH has never been developed, and thus lyophilized preparations are common which are dissolved prior to injection.
As treatment with hGH to alleviate dwarfism takes a long period of years, self-injection is allowed and generally conducted at home from the start of its administration. When using a lyophilized preparation of hGH, the preparation is dissolved in an attached solvent and then injected subcutaneously or intramuscularly by the patient himself (usually a child) or his family members. Thus, it is the patient or his family members that carry out the dissolution procedure of the lyophilized preparation. Therefore, it is necessary for a physician in charge to give an adequate guidance as to how to dissolve it in order to avoid formation of aggregation products which leads to reduction of biological activity of the hGH. Their product inserts also contain cautions and instructions that hGH should be dissolved with a gentle circular motion.
As aggregates formation has also been noted in production steps of lyophilized preparations, various attempts have been made to suppress aggregates formation. However, there still are eager needs for development of more easily handled, stable preparations than the usual types of preparations which are dissolved prior to use. Recently, a kit type preparation with an associated syringe has come into use. But it has a complex structure so that dissolution of the lyophilized hGH is effected within the syringe, and it therefore makes it necessary to give especially careful and thorough explanation to the patient or his family on how to use it. Also, risks of unforeseeable erroneous handling cannot be cleared off.
As hGH is thus commonly injected at home by the patient or his family, provision of an aqueous form hGH preparation, in which dissolution procedure is eliminated, would promote convenience. Such a proper form of aqueous preparation would serve to ease the burden imposed on the patient and his family because it can be handled easily without the need of structurally complex devices employed in usual two-chamber type products requiring dissolution prior to use, e.g., a pen-type product including lyophilized hGH and a solvent which are separated by a partition.
There are following predominant patent applications addressed to hGH aqueous preparations.
CABI, in Unexamined Patent Publication No. 508156/1994 (hereinafter referred to as "CABI publication"), discloses an injectable composition of hGH or its active analogues with a pH of 5-7.5 and containing 2-50 mM citric acid as a buffering agent. It is stated in the same publication that better stability has been obtained by employing citrate than phosphate and that pH of about 6.0-7.0 is relatively preferable.
The above CABI publication teaches that the preparation set forth therein is stable for at least 12 months. In the same publication, however, "being stable" with regard to the monomer is defined as keeping the content not less than 85% of initial. Considering that specifications on the monomer content is generally considered to be not less than 90%, that preparation by CABI hardly seems to have sufficient stability.
On the other hand, through studies for improvement of quality and stability, the present inventors have also found, separately from the above disclosure by CABI, that pH is a crucial factor in the production of hGH preparation in the form of a solution, that use of a buffering agent which can maintain the preferable pH of 5-7, more preferable pH of 5.5-6.5, and that citrate, for example, is effective as such a buffering agent (Unexamined Patent Publication No. 92125/1996).
On the other hand, Genentech Inc. describes, in Unexamined Patent Publication No. 509719/1995 (hereinafter referred to as the "Genentech publication"), a liquid form hGH preparation comprising hGH, mannitol, a buffer and a nonionic surfactant. Citrate buffer is exemplified in that publication as being preferable.
Further, in Unexamined Patent Publication No. 507497/1993, Novo Nordisk Pharma describes a preparation which is produced first by crystallizing hGH by addition of acetone or ethanol in the presence of a divalent cation, e.g. Zn.sup.2+, lyophilizing the crystals and putting the dried crystals into a pH 6.1-6.2 suspension comprising, e.g., phosphate, zinc acetate, glycerol, and benzylalcohol. In that publication, 6-month stability tests results at 22-24.degree. C. are reported for the suspension made of hGH crystals, using ion-exchange HPLC for patterns of deamidation and decomposition and GPC for content of dimers and polymers, respectively. It is reported that even after 6 months the content of desamide products in the preparation was 5.0%, didesamide products 1.8%, dimers 1.2% and polymers 0.3% preparation, any of which were lower than the stability test results with the solutions reconstructed from usual lyophilized preparations. The same publication states that Zn.sup.2+ is essential in crystallization to obtain large crystals. In addition, in spite that a water-soluble solvent such as acetone or ethanol is required in the crystallization of hGH, no mention is given to any alteration in secondary or higher structures of hGH crystals in the suspension, thus leaving points unclarified.
Acetone and ethanol are often used in purification of proteins to obtain them in precipitation. This method makes use of the lower solubility of proteins to organic solvents. Though the concentration of such organic solvents in the same publication are lower than those used for making proteins precipitate, they would not be preferable for a pharmaceutical preparation administered to a human.
Upon this background, the present inventors pursued further research for an improved aqueous hGH preparation. In the process of our research, a variety of aqueous hGH preparations in liquid state were made using citrate buffer in accordance with what is described in the above unexamined patent publication by the present inventors and the CABI publication, and tests were conducted for their stability for a variety of time periods. It was then noted that any of these preparations developed slightly visible fine particles which were distinguished from aggregates. The fine particles were removable with a 0.22 .mu.m filter, for example, but could be found again after a long-term storage or under such a stress as shaking. As formation of such fine particles would be problematic in quality with a pharmaceutical product, development of a new aqueous preparation was needed in which formation of such particles is suppressed.
On the other hand, while citrates are widely used in injectable preparations as buffering agents in slightly to weakly acidic conditions, it has been reported that they cause pain when the solution is injected subcutaneously or intramuscularly as is the case of the hGH (Unexamined Patent Publication No. 510031/94). The present inventors themselves also tested the preparations produced in accordance with the descriptions in the above CABI publication and above Unexamined Patent Publications by the present inventors, and found that these citrate-containing hGH injections cause substantial pain when the solution is infused.
Unlike stability problems of a preparation, the pain upon infusion is not a significant problem from the viewpoint of quality of the preparation. However, considering that a hGH preparation is injected on a frequent basis for a log period of time and that the patients of interest are children, and in order to ease the pain in the patients and, at the same time, thereby assuring compliance, it naturally is desired that the injectable solution itself would cause no pain. There is so far no preparation actually used in treatment and that causes no pain in the patients upon infusion of the composition. Therefore, in light of pain upon infusion, further room for an improvement is left in any of the preparations described in the above CABI publication and the Genentech publication (the latter describes citrate buffer as being preferable), as well as in the preparation according to the above patent application by the present inventors. Elimination or reduction of pain would be beneficial to the patients.
The first objective of the present invention is to solve the above stability problems of a hGH aqueous preparation for injection, i.e., to provide a stable hGH aqueous preparation in which deamination, polymerization and aggregation are sufficiently suppressed and formation of the above fine particle is also suppressed.
Another objective of the present invention is provide a hGH aqueous preparation with which pain due to its composition felt during infusion in subcutaneous or intramuscular injection is eliminated or reduced.
With a variety of hGH preparations unintentionally kept in storage before lyophilization, the present inventors found that preparations obtained by dissolving hGH in aqueous solutions the pH of which was maintained at 6 with maleate buffer or succinate buffer are comparably stable to the solution (described in Unexamined Patent Publication No. 92125/1996) in which the same pH was kept by means of citrate buffer, and that they do not produce dimers or polymers, nor do they produce deamidated products. The tests conducted and best reflecting changes in the quality of human growth hormone were: determination of monomer content on size-elimination high performance liquid chromatography (SE-HPLC), determination of the content of deamination products using high-performance liquid chromatography using a reverse-phase column, observation of general appearance and pH measurement. As a result of further intense examinations on the aqueous preparations during production, under conditions with thermal stress and after 6-month storage in cool place, maleate buffer, succinate buffer and citrate buffer were selected as having proper buffering ability out of pyruvate buffer, acetate buffer, phosphate buffer, citrate buffer, succinate buffer and maleate buffer.
It was confirmed again that by employing those proper buffers stabilization of hGH could be achieved at slightly or weakly acidic pH. However, closer observation revealed that slightly visible fine particles which scatter light and are distinguished from aggregates were detectable when preparations had been made under slightly to weakly acidic conditions. Therefore, we examined the effect of a number of compounds in search of a method to suppress the formation of the fine particles. As a results, we discovered that a low concentration of benzalkonium chloride can effectively suppress the formation of the fine particles. Further studies were carried out on the basis of this finding and it was made clear that a stable hGH aqueous preparation, in which deamination, polymerization and aggregation, as well the fine particle formation is suppressed, can be produced by employing certain formulations of aqueous preparation according to which hGH is dissolved in a solution adjusted to slightly to weakly acidic pH and containing benzalkonium chloride. The present invention was thus completed.
Meanwhile, further studies of such slightly to weakly acidic aqueous preparations led to an unexpected finding that while citrate used as a buffering agent for maintaining this pH range caused substantial pain in subcutaneous injection when the solution was infused, maleate or succinate, which are similar polycarboxylic acid salts, in contrast caused no pain substantially. On the basis of this finding, a preferable hGH-containing aqueous pharmaceutical composition has been successfully prepared which has good stability and causes no pain upon infusion.